Polypeptides and polynucleotides for enhancing immune reactivity to HER-2 protein

a technology of polynucleotide and immune reactivity, which is applied in the field of polypeptides and polynucleotides for enhancing immune reactivity to her2 protein, can solve the problems of skin sensitivity or itchiness, interference with the immune system, and surgery alone cannot eliminate cancer

Inactive Publication Date: 2006-06-13
OHIO STATE INNOVATION FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0025]The present invention also relates to an immunogenic composition containing the chimeric HER-2 B cell peptide or multivalent HER-2 B cell peptide and a pharmacologically acceptable carrier. The preferred carrier is a biodegradeable microsphere. Such immunogenic compositions are useful for treating or preventing malignancies with which overexpression of the HER-2 protein is associated.
[0026]The present invention also relates to polynucleotides which encode at least one of the HER-2 B cell epitopes described above. Such polynucleotides are useful for producing the epitope by recombinant techniques. The present invention also relates to isolated polynucleotides having a sequence which encodes a chimeric HER-2 B cell peptide of the present invention. Such polynucleotides are useful for preparing the chimeric HER-2 B cell peptide. Such polynucleotides are also useful in an immunogenic composition (e.g., DNA vaccine) for treating or preventing malignancies in which overexpression of the HER-2 protein is associated. Preferably, such immunogenic compositions are administered intramuscularly.
[0033]The present invention also encompasses isolated polynucleotides which encode one or more of the HER-2 CTL epitopes described above. Such polynucleotides are useful for producing the epitope by recombinant techniques. The present invention also encompasses isolated polynucleotides having a sequence which encodes a chimeric HER-2 CTL peptide. Such polynucleotides are useful for preparing the chimeric CTL cell epitope peptide. Such polynucleotides are also useful in an immunogenic composition, (e.g., DNA vaccine) for treating or preventing malignancies in which the HER-2 oncogene is associated.

Problems solved by technology

Unless the cancer is restricted to a defined area, surgery alone cannot eliminate the cancer.
The side effects of such treatment include skin sensitivity or itchiness, interference with the immune system, sometimes queasiness and, rarely, radiation fibrosis where an affected portion of the lung becomes fibrous.
Since this is not a perfectly selective system, normal cells are affected as well.
Negative side effects include nausea, tiredness, loss of appetite, hair loss and diarrhea.
Overexpression of HER-2 protein is correlated with a poor prognosis in both breast and ovarian cancer.
Immunization of BDIX rats with the same immunogen, however, did not result in antibody response nor did it inhibit the growth of syngeneic neu-expressing B 104 neuroblastoma cells, suggesting that this strategy was insufficient to induce immune responses in the rat.
Despite the results of the studies described above, it is still uncertain whether effective immune responses can be generated in humans using cell-or protein-based vaccine strategies targeting HER-2 or the HER-2 ECD, as HER-2 is a non-mutated, “self” antigen.

Method used

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  • Polypeptides and polynucleotides for enhancing immune reactivity to HER-2 protein
  • Polypeptides and polynucleotides for enhancing immune reactivity to HER-2 protein
  • Polypeptides and polynucleotides for enhancing immune reactivity to HER-2 protein

Examples

Experimental program
Comparison scheme
Effect test

example 1

Peptide DWI MVF; HER-2 (376–395) MVF

[0092]The epitope, named DW1, comprises amino acids 376–291 of the HER-2 protein linked to the promiscuous Th cell epitope MVF. DW1 is predicted to be α-helical with a slight turn propensity.

[0093]Synthesis: The 20 amino acid HER-2 sequence was attached to the N-terminus of MVF 288–302 by the four amino acid linked sequence Gly-Pro-Ser-Leu SEQ ID NO. 20. The resulting peptide was named DW1MVF indicating DW1 placement at the N-terminus as opposed to MVFDW1 which would represent C-terminal position. The first amino acid was joined manually and completion monitored by Kaiser ninhydrin test. Subsequent couplings were performed on the Milligen / Biosearch 9600 peptide synthesizer. After final deblocking the peptide was cleaved from the resin with Reagent PU. An extended cleavage time is necessary since the peptide contains Arg-2,2,5,7,8-pentamethylchroman-6-sulfonyl(PMC) and His. (A yellow cleavage solution is observed when histidine is present).

[0094]Pu...

example 2

Peptide MVFDW4: HER-2 (628–647) MVF

[0096]MVFDW4 comprises an altered sequence of the peptide extending from amino acid 628 through 647 of the HER-2 protein. The native sequence contains 3 cysteine residues whose disulfide bonding pairs are unknown. Since the cysteines at position 634 and 642 had the potential to form a bridge, Cys 630 was substituted with Gly. Substituting glycine for cysteine is one way to preserve the relative size of the R group at that position. Synthesis proceeded by first making the DW4 (628–647) peptide attached to the linker then extending the sequence N-terminally by addition of the NWF (288–302) T helper cell sequence. This produced the MVFDW4 peptide.

[0097]In order to create the disulfide bond, the tBut protecting group was cleaved giving the free thiol form. The mercuric acetate / 2-mercaptoethanol procedure reduces production of disulfide bonded multimers. Analytical HPLCs of the crude product and samples were compared. In the crude sample, two sharp peak...

example 3

Peptide DW5MVF; HER-2 (115–136) MVF

[0100]Synthesis: MVF 288–302 plus the four residue amino acid linker was connected to the resin as described above in example 1 and the sequence continued with amino acids 115–136 of the HER-2 protein. This produced the peptide DW5MVF. The sequence is predicted to be a β-turn with high aggregation potential. This necessitated double coupling critical residues A115, V116, T127, V129 and S133.

[0101]Purification and characterization: DW5MVF was cleaved and extracted with ether and water. Extraction was quite difficult as the peptide formed dense, sticky aggregates which were only minimally soluble by addition of acetic acid. Analytical HPLC of the crude sample showed one predominant peak with a minor doublet. Semipreparative HPLC was used to separate the doublet. The lyophilized sample was readily dissolved in dilute acetic acid for analytical HPLC. A sample was subjected to time of flight mass spectrometry and yielded a molecule of the correct molecu...

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Abstract

Compositions for stimulating the immune system and for treating malignancies associated with overexpression of the HER-2 protein are provided. Such compositions include immunogenic epitopes of the HER-2 proteins and chimeric and multivalent peptides which comprise such epitopes. The present invention also relates to polynucleotides which encode the chimeric peptides. Also provided are pharmaceutical compositions comprising such immunogenic compositions. Methods for stimulating an immune response to HER-2 protein are provided. Methods for treating breast cancer, ovarian cancer, prostate cancer, colon cancer and lung cancer are provided.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]Under 35 USC §119(e)(1), this application claims the benefit of prior U.S. provisional application 60 / 146,869, filed Aug. 3, 1999.[0002]The work described in this application was supported, at least in part, by grants PHS / NIH P30 CA-16058 and NIH / NCI RO1 CA 84356-01A1 from the National Cancer Institute. The United States government has certain rights in this invention.BACKGROUND[0003]Currently, the most common forms of treating breast cancer involve surgery, chemical intervention, and / or radiotherapy. Unless the cancer is restricted to a defined area, surgery alone cannot eliminate the cancer. Accordingly, radiation treatment is often given after surgery to destroy cancer cells that are near the surgical site and that have evaded surgery. The side effects of such treatment include skin sensitivity or itchiness, interference with the immune system, sometimes queasiness and, rarely, radiation fibrosis where an affected portion of the lung be...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): A61K39/00C12N15/09A61K9/00A61K9/107A61K9/16A61K47/06A61K47/34A61P35/00A61P37/02C07K7/06C07K7/08C07K14/71C07K14/82C07K16/46C07K19/00
CPCA61K9/0019A61K9/1647C07K14/71A61K2039/64A61K2039/6037A61P35/00A61P37/02A61P37/04
Inventor KAUMAYA, PRAVIN T. P.STEVENS, VERNON C.TRIOZZI, PIERRE L.
Owner OHIO STATE INNOVATION FOUND
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